[논문]Cytochrome <tex>$C_{550}$</tex> is Related to Initiation of Sporulation in Bacillus subtilis

Shin Inji; Ryu Han-Bong; Yim Hyung-Soon; Kang Sa-Ouk

Cytochrome $C_{550}$ is Related to Initiation of Sporulation in Bacillus subtilis 원문보기

The journal of microbiology, v.43 no.3, 2005년, pp.244 - 250

Shin Inji (Laboratory of Biophysics, School of Biological Sciences, and Institute of Microbiology, Seoul National University) , Ryu Han-Bong (Laboratory of Biophysics, School of Biological Sciences, and Institute of Microbiology, Seoul National University) , Yim Hyung-Soon (Laboratory of Biophysics, School of Biological Sciences, and Institute of Microbiology, Seoul National University) , Kang Sa-Ouk (Laboratory of Biophysics, School of Biological Sciences, and Institute of Microbiology, Seoul National University)

Abstract ▼ AI-Helper

The effect of cytochrome $c_{550}$ encoded by cccA in Bacillus subtilis during the event of sporulation was investigated. The sporulation of cccA-overexpressing mutant was significantly accelerated, while disruptant strain showed delayed sporulation in spite of the same growth rate. Activity of sporulation stage-0-specific enzyme, extracellular $\alpha-amylase$ of mutant strains was similar to that of the control strain, but cccA-overexpressing mutant exhibited higher activity of stage-II-specific alkaline phosphatase and stage-III-specific glucose dehydrogenase when compared to deletion mutant and control strain. Northern blot analysis also revealed that cccA-overexpressing mutant showed high level of spo0A transcripts, while the disruptant rarely expressed spo0A. These results suggested that although cytochrome $c_{550}$ is dispensable for growth and sporulation, expression of cccA may play an important role for initiation of sporulation through regulation of spo0A expression.

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제안 방법

To analyze the possible role of cccA in sporulation, its overexpression vector and plasmid for disruption were constructed, The full length cccA was amplified by PCR by using primers, 5'-GGTATATGGATAAGCTTCCT- TTATTTTACTGAAAAATGATGTC-3' and 3'-CCC- ACAGTTTTTAATTTATTTTCCTAGGAAAAAGAG- 5' (the underlined sequences indicate HinHU and BamHI site, respectively) and was ligated with pGEM-T Easy. The resultant plasmid, pGEM-cccA, was transformed into E.
To investigate further as how cytochrome c₅₅₀ can activate sporulation, transcripts level of spore specific gene, spo0A was analyzed. Because spo0A is expressed at the initiation of sporulation and accumulation of spo0A to a critical concentration is required for the commitment of cells to the sporulating pathway (Hoch, 1993; Spiegelman et al.

이론/모형

Total RNA isolation from B. subtilis mutants was performed by using 'Modified Kirby Mix', designed by Van Dessel et al. (2004). Cells were homogenized and dis- mpted by Modified Kirby Mix, which contained 1% N- lauroylsarcosine, 6% p-aminosalicylic acid sodium salt, and 6% phenol in 0.

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